RESUMO
Autophagy is the major cellular process of degradation and is modulated by several signaling pathways. Phosphatidylinositol 3-kinase (PtdIns3K) class III (Vps34) and PtdIns3K class I regulate the autophagy pathway positively and negatively, respectively. Both classes of PtdIns3K participate in the synthesis of phosphatidylinositol 3-phosphate (PtdIns3P), which plays a crucial role in autophagosome biogenesis and membrane traffic. PtdIns3P is a membrane phospholipid that is associated with endogenous FYVE domain-containing proteins. Indeed, such interactions facilitate autophagosome fusion with lysosomes and subsequent cargo degradation. During starvation-induced autophagy, the expression of FYVE domain-containing proteins increases, and their binding to PtdIns3P is strengthened. Nonetheless, not all FYVE domain proteins are related to the induction of autophagy. This method report presents the quantification of PtdIns3P synthesis by using cells either transiently transfected with or stably expressing FYVE-dsRed.
Assuntos
Autofagia , Microscopia de Fluorescência/métodos , Biologia Molecular/métodos , Fosfatos de Fosfatidilinositol/biossíntese , Proteínas Recombinantes/metabolismo , Linhagem Celular , Corantes Fluorescentes/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fosfatos de Fosfatidilinositol/análise , Proteínas Recombinantes/análise , Proteínas Recombinantes/genéticaRESUMO
Macroautophagy (usually referred to as autophagy) is the most important degradation system in mammalian cells. It is responsible for the elimination of protein aggregates, organelles, and other cellular content. During autophagy, these materials (i.e., cargo) must be engulfed by a double-membrane structure called an autophagosome, which delivers the cargo to the lysosome to complete its degradation. Autophagy is a very dynamic pathway called autophagic flux. The process involves all the steps that are implicated in cargo degradation from autophagosome formation. There are several techniques to monitor autophagic flux. Among them, the method most used experimentally to assess autophagy is the detection of LC3 protein processing and p62 degradation by Western blotting. In this chapter, we provide a detailed and straightforward protocol for this purpose in cultured mammalian cells, including a brief set of notes concerning problems associated with the Western-blotting detection of LC3 and p62.
Assuntos
Autofagia/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Biologia Molecular/métodos , Animais , Western Blotting , Células Cultivadas , Eletroforese/métodos , Fibroblastos/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/análise , Proteína Sequestossoma-1/metabolismoRESUMO
Mitochondria-associated membranes (MAMs) are structures that regulate physiological functions between endoplasmic reticulum (ER) and mitochondria in order to maintain calcium signaling and mitochondrial biogenesis. Several proteins located in MAMs, including those encoded by PARK genes and some of neurodegeneration-related proteins (huntingtin, presenilin, etc.), ensure this regulation. In this regard, MAM alteration is associated with neurodegenerative diseases such as Parkinson's (PD), Alzheimer's (AD), and Huntington's diseases (HD) and contributes to the appearance of the pathogenesis features, i.e., autophagy dysregulation, mitochondrial dysfunction, oxidative stress, and lately, neuronal death. Moreover,, ER stress and/or damaged mitochondria can be the cause of these disruptions. Therefore, ER-mitochondria contact structure and function are crucial to multiple cellular processes. This review is focused on the molecular interaction between ER and mitochondria indispensable to MAM formation and on MAM alteration-induced etiology of neurodegenerative diseases.
Assuntos
Morte Celular/fisiologia , Retículo Endoplasmático/metabolismo , Membranas Mitocondriais/metabolismo , Estresse Oxidativo/fisiologia , Doença de Parkinson/metabolismo , Animais , Humanos , Mitocôndrias/metabolismo , Biogênese de OrganelasRESUMO
Pompe disease or glycogen storage disease type II (OMIM: 232300) is a lysosomal storage disorder resulting from a partial or total lack of acid alphaglucosidase, which may produce muscle weakness, gait abnormalities, or even death by respiratory failure. In the last decade, autophagy has been proposed as a mechanism involved in the severity of symptoms related to this disorder and as a potential therapeutic target to alleviate disease progression. This review summarizes the relationship between autophagy and Pompe disease, including what information has been recently discovered and what remains unclear.
Assuntos
Autofagia , Doença de Depósito de Glicogênio Tipo II/patologia , Animais , Modelos Animais de Doenças , Terapia de Reposição de Enzimas , Doença de Depósito de Glicogênio Tipo II/metabolismo , Doença de Depósito de Glicogênio Tipo II/terapia , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , alfa-Glucosidases/genética , alfa-Glucosidases/metabolismoRESUMO
Arginase activity was measured in serum and biopsy from healthy individuals and colorectal cancer patients. Arginase activity in tumor samples (87 +/- 7.7 U/g tissue) was significantly higher than in controls (40.7 +/- 3.3 U/g tissue). However, serum arginase activity did not show any significant change in both groups. Finally, the micromethod used to quantify arginase activity in this study is superior to other methods because it has increased sensitivity, requires less sample, and is less time-consuming. Arginase differences are significant, according to the t-test (P<0.05)
Assuntos
Arginase/sangue , Carcinoma/metabolismo , Neoplasias Colorretais/metabolismo , Bioensaio , Biomarcadores Tumorais , Biópsia , Carcinoma/diagnóstico , Neoplasias Colorretais/diagnóstico , Estabilidade Enzimática , Humanos , Sensibilidade e Especificidade , Ureia/metabolismoRESUMO
Exposure of cerebellar granule cells to 1-methyl-4-phenylpiridinium (MPP(+)) results in cell death. We have studied the implication of various membrane transporter systems on MPP(+) neurotoxicity, including the dopamine transporter system (DAT) and cationic amino acid transporters (CAT). We have showed a partial protection against MPP(+) toxicity when the dopamine transporter is inhibited by 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]4-(3-phenylpropyl)piperazinedihydrochloride (GBR-12909). However, almost full protection is only achieved by the simultaneous addition of GBR-12909 and cationic amino acids. These results suggest two ways system of MPP(+) entrance into cerebellar granule cells: the DAT with high activity and the CAT with low activity. We also demonstrated that 5,7-dichlorokynurenic acid (MK-801) failed to protect against MPP(+) exposure, evidencing that N-methyl-D-aspartate (NMDA) receptor is not involved in the MPP(+)-induced cell death.
Assuntos
1-Metil-4-fenilpiridínio/metabolismo , Proteínas de Transporte/efeitos dos fármacos , Córtex Cerebelar/efeitos dos fármacos , Herbicidas/metabolismo , Intoxicação por MPTP/metabolismo , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso , Neurônios/efeitos dos fármacos , Neurotoxinas/metabolismo , 1-Metil-4-fenilpiridínio/toxicidade , Sistemas de Transporte de Aminoácidos Básicos/efeitos dos fármacos , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Aminoácidos Básicos/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas de Transporte/metabolismo , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Córtex Cerebelar/metabolismo , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Inibidores da Captação de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas/fisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Herbicidas/toxicidade , Intoxicação por MPTP/fisiopatologia , Masculino , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Neurônios/metabolismo , Neurotoxinas/toxicidade , Piperazinas/farmacologia , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/efeitos dos fármacosRESUMO
Lithium protects cerebellar granule cells from apoptosis induced by low potassium, and also from other apoptotic stimuli. However, the precise mechanism by which this occurs is not understood. When cerebellar granule cells were switched to low potassium medium, the activation of caspase 3 was detected within 6 h, suggesting a role of caspase 3 in mediating apoptosis under conditions of low potassium. In the same conditions, lithium (5 mM) inhibited the activation of caspase 3 induced by low potassium. As lithium did not inhibit caspase 3 activity in vitro, these results suggest that this ion inhibits an upstream component that is required for caspase 3 activation. Lithium is known to inhibit a kinase termed glycogen sythase kinase 3 (GSK3), which is implicated in the survival pathway of phosphatidylinositol 3-kinase/protein kinase B (PI3K/PKB). Here we demonstrate that low potassium in the absence of lithium induces the dephosphorylation, and therefore the activation, of GSK3. However, when lithium was present, GSK3 remained phosphorylated at the same level as observed under conditions of high potassium. Low potassium induced the dephosphorylation and inactivation of PKB, whereas when lithium was present PKB was not dephosphorylated. Our results allow us to propose a new hypothesis about the action mechanism of lithium, this ion could inhibit a serine-threonine phosphatase induced by potassium deprivation.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Caspases/metabolismo , Cerebelo/metabolismo , Lítio/farmacologia , Neurônios/metabolismo , Deficiência de Potássio/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Animais , Caspase 3 , Células Cultivadas , Cerebelo/efeitos dos fármacos , Cerebelo/patologia , Ativação Enzimática/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase , Fosforilação/efeitos dos fármacos , Deficiência de Potássio/patologia , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos WistarRESUMO
Acute treatment with valproate and Li+ was found to protect cultured cerebellar granule cells against apoptosis induced by low K+ (5 mM). Because the protection was unaffected by MK801 (N-methyl-D-aspartate receptor inhibitor), an increase in glutamate release cannot be responsible for the observed neuroprotection. Insulin also protects against low-K+-induced apoptosis of cerebellar granule cells. This protection is totally dependent on LY294002 (a phosphatidylinositol 3-kinase inhibitor). These results suggest a role for phosphatidylinositol 3-kinase in the neuroprotection induced by insulin. Likewise, and in contrast with the results observed with Li+, the protection induced by valproate is also dependent on insulin and LY294002. Moreover, valproate (a branched-chain fatty acid) does not change the plasma membrane microviscosity under physiological conditions. These results suggest that valproate protects against low-K+-induced apoptosis by acting in the phosphatidylinositol 3-kinase/protein kinase B pathway. The protection by Li+ is independent of this transduction pathway.